Improved detection of mutations in the p53 gene in human tumors as single-stranded conformation polymorphs and double-stranded heteroduplex DNA.

Abstract

Molecular Biology of Breast Cancer Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037 Research in the last 3 years has made it increasingly clear that a large variety of human tumors contain mutated p53 tumor suppressor genes (reviewed in ref. 1). Since mutations are most often distributed over at least 5 exons (exons 5-9) spanning 2.9 kb of coding sequence, faster methods for scanning the region have been developed. ~2) A popular method for detection of point mutations and deletions in the p53 gene is single-stranded conformation polymorphism (SSCP). (2'3) The technique is based on altered migration speeds through solid supports of singlestranded DNA fragments carrying mutations. The altered migration of DNA is presumably caused by differences in the conformation of single-stranded DNA. The method depends heavily on experimental conditions that optimize migration differences of the conformation polymorphs. Thus, adding glycerol to the polyacrylamide, reducing the temperature, increasing the length of the run, and so forth result in a greater level of reproducibility. ~2'3~ In spite of these modifications, reproducibility and resolution present recurrent problems using polyacrylamide as a gel support, presenting the need for novel polymers for this purpose. There is also a need to develop a simple nonradioactive technique for screening for mutations in the p53 gene in the clinical laboratory. A gel electrophoresis technique that takes advantage of differences in mobility of wild-type and mutation-bearing DNA fragments could prove useful for this purpose. Double-stranded DNA as heteroduplexes (HTX) migrate at different rates compared to DNA as homoduplexes in solid supports. (4's~ These differences can be visualized easily by staining with ethidium bromide. However, the resolution ability of polyacrylamide gel supports for detection of the two species varies with the sequence of the DNA fragments. ~4's~ In an effort to improve the sensitivity levels of the radioactive SSCP analysis, and to resolve the nonradioactive duplexes, we began to experiment with other polymers. We tested polyacrylamide, Hydrolink-D5000, and HydrolinkMDE under identical experimental conditions with and without 10% glycerol. We found that Hydrolink-MDE (AT Biochem, Malvern, PA), a vinyl polymer, gave improved resolution of bands as well as reproducibility in the detection of both single-stranded DNA in SSCP and of DNA duplexes in the HTX analyses. Because the SSCP analysis is unable to detect mutations that do not result in polymorphs with altered migrations, a combination of SSCP analysis with HTX analysis might provide more reliable results than either one alone. Human mammary tumors were analyzed by SSCP for mutations in p53 exons 5-9 using 5% polyacrylamide. ~6) A cell line derived from normal breast tissue (HBL-100) was used as a negative control, whereas breast tumor cell lines served as positive controls (SKBR3 and HS578 for exon 5, T47D for exon 6, MW for exon 7, and BT474 for exon 8). The colon cancer cell line, SW480 was used as a positive control for mutation in exon 9 of the p53 gene. It should be noted that these cell lines, carrying mutations in the various exons of the p53 gene, have lost the normal allele of the p53 gene and are therefore, hemior homozygous mutants. Primary breast tumor DNAs were also analyzed by SSCP on polyacrylamide and it was found that 10 out of 59 (17%) primary tumors had mutations in this region of the p53 locus. (6) Then, DNA from the same panel of cell lines as well as primary tumors considered to be questionable positives by SSCP analysis on polyacrylamide gels (PGE) was retested with the polymer, Hydrolink-MDE (MDE), a modified polyacrylamide-based vinyl polymer. Representative results are shown in Figure 1 for exon 6 of the p53 gene, as the majority of mutations observed in breast carcinomas were detected in these sequences. It should be noted that the sharp contrast in resolution efficiency seen in PGE versus MDE is reproducible, but is peculiar to exon 6, since the other fragments (exons 5, 7, 8, 9) showed comparable separations in the two gel matrices (shown for exon 8 in Fig. 2). As seen in Figure 1, there is improved resolution of several of the conformation polymorphs in MDE in comparison to PGE. As a result, alterations in migration in the DNA fragments caused by mutations are more easily discernible (compare 4813, 5600, and 4805 in MDE versus PGE). Because single-stranded DNA mobility is also subject to environmental and gel conditions, some mobility differences are questionable with the new polymer (Fig. 1, 5633). Tumors 4811 and 5594 are negative for mutations in exon 6 (Fig. 1), but showed mobility shifts compared to HBL-100 in exons 8 and 9, respectively,